Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play many important physiological roles. nAChRs naturally exist as a family of diverse subtypes. Each nAChR subtype is a pentamer and is defined by its composition of subunits, which in mammals are products of sixteen different genes. Each nAChR subtype has a distinctive distribution and set of characteristics. nAChR subtype diversity in the brain is physiologically important and provides opportunities for target-based drug treatment of neuropsychiatric conditions. Of particular interest in this project, native nAChR subtypes containing a6, b2, b3 a4 subunits (a6*-nAChR) are expressed in midbrain, dopaminergic, pleasure and reward centers. This means that drugs selectively targeting a6*-nAChR could be used to treat nicotine or other drug dependence, and/or Parkinson's disease. However, characterization of these a6*-nAChR subtypes in isolation has been elusive, in part because they are naturally expressed over a background of other nAChR subtypes, but also because of difficulties during heterologous expression in precisely controlling subunit composition, stoichiometry, and arrangement, perhaps due to the absence of chaperone-guided processes. We seek to overcome these obstacles toward a6*-nAChR characterization and heterologous expression using tethered pentameric subunit constructs. We will begin to test the hypothesis that nAChR construction from tethered subunits enforces correct subunit assembly in the absence of neuron-specific chaperone proteins, preserving properties of native nAChR subtypes. The specific aims are to heterologously express and characterize, first in Xenopus oocytes and then in a mammalian cell host, a6*-nAChR composed of defined, tethered subunits. These subunits will be created by systematically introducing a6 and b3 subunits into a construct proven to encode fully functional nAChR. Standard assays will be used to confirm construct expression from message to functional a6*-nAChR and to characterize functional a6*-nAChR. As befits an R21 project, successful heterologous expression of covalently-linked a6b2b3- and a6a4b2b3- nAChR constructs will provide proof of concept for expression of other, complex nAChR subtypes using the tethered pentamer approach. No other approach can ensure the subunit composition, stoichiometry and arrangement that define a given nAChR subtype. Pure, plentiful expression of a6*-nAChR as tethered pentamers would enable unambiguous determination of subunit contributions to subtype function, and tests of subtype selectivity of drugs at these targets. Of public health relevance, these advances could extend to improved treatment of diseases ranging from addictions to movement disorders. PUBLIC HEALTH RELEVANCE: Nicotinic acetylcholine receptors (nAChRs) containing the a6 subunit are implicated in diseases ranging from addictions to Parkinson's disease. These receptors have proved difficult to study using natural sources, due to a combination of scarcity and contamination with other nAChR subtypes. Artificial expression of a6-containing nAChRs would provide a valuable alternative, but obtaining appropriate expression of these complex receptors has been extremely challenging. We propose an approach using linked receptor subunits to make a6- containing nAChRs with properties matching those of their naturally-expressed counterparts, as an alternative to the usual association of individual subunits. This will enforce desired subunit interactions, facilitating expression in a variety of experimentally amenable systems. In turn, this will greatly simplify studies of these important receptors, enhancing knowledge of their properties and helping to guide development of nAChR- directed treatments for addictions and Parkinson's disease.